HIV related peptides

ABSTRACT

This invention is the discovery of novel specific epitopes and antibodies associated with long term survival of HIV-1 infections. These epitopes and antibodies have use in preparing vaccines for preventing HIV-1 infection or for controlling progression to AIDS.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/115,430 filed on Jan. 11, 1999 and to U.S. ProvisionalApplication Ser. No. 60/132,760 filed on May 6, 1999 each of which isincorporated by reference herein.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not Applicable

BACKGROUND OF THE INVENTION

Efforts to develop a protective human immunodeficiency virus-1 (HIV-1)vaccine have been hindered by difficulties in identifying epitopescapable of inducing broad neutralizing antibody responses. The highmutation rate that occurs in HIV-1 envelope proteins and the complexstructure of gp120 as an oligomer associated with gp41 results in a highdegree of antigenic polymorphism. To overcome these obstacles, we haveidentified antigenic and immunogenic mimics of HIV-1 epitopes that arespecific to HIV-resistant individuals. The epitope mimics wereidentified by screening random peptide libraries using sera fromHIV-infected subjects who were long term non-progressors (LTNPs). Afterextensive counter-screening with HIV-negative sera, we isolated peptidesspecifically recognized by antibodies from HIV-1 infected individuals.

SUMMARY OF THE INVENTION

The present invention includes an antigenic peptide of less than 100amino acids having an antigenic subsequence selected from the groupconsisting of X-KSSGKLISL-X (SEQ ID NO:1), X-CNGRLYCGP-X (SEQ IDNO:2)and X-GTKLVCFAA-X (SEQ ID NO:3), wherein X is independently anamino acid or sequence of amino acids with the proviso that X is notidentical to the amino acid or amino acids naturally flanking thecorresponding subsequences in HIV-1.

An additional embodiment of this invention is a vaccine for protectingagainst HIV-1 infection comprising an antigenic peptide of less than 100amino acids having an antigenic subsequence selected from the groupconsisting of X-KSSGKLISL-X (SEQ ID NO:1), X—CNGRLYCGP-X (SEQ ID NO:2)and X-GTKLVCFAA-X (SEQ ID NO:3), wherein X is independently an aminoacid or sequence of amino acids with the proviso that X is not identicalto the amino acid or amino acids naturally flanking the subsequences inHIV-1.

Embodiments of the present invention also include a composition forraising antibodies against HIV-1, said composition comprising anantigenic determinant selected from the group consisting of KSSGKLISL(SEQ ID NO:4), CNGRLYCGP (SEQ ID NO:5) and GTKLVCFAA (SEQ ID NO:6),wherein the composition does not give rise to HIV-specific antibodies tomore than eight other antigenic determinants on HIV-1.

A further embodiment of this invention is a vaccine for protectingagainst HIV-1, said vaccine comprising an antigenic determinant selectedfrom the group consisting of KSSGKLISL (SEQ ID NO:4), CNGRLYCGP (SEQ IDNO:5) and GTKLVCFAA (SEQ ID NO:6), wherein the composition does not giverise to HIV-specific antibodies to more than eight other antigenicdeterminants on HIV-1.

The present invention also encompasses a method for raising antibodiesagainst HIV-1, said method comprising administering to an animalcompetent to raise antibodies an amount of a composition comprising anantigenic determinant selected from the group consisting of KSSGKLISL(SEQ ID NO:4), CNGRLYCGP (SEQ ID NO:5) and GTKLVCFAA (SEQ ID NO:6),wherein the composition does not give rise to HIV-specific antibodies tomore than eight other antigenic determinants on HIV-1, said amountsufficient to raise antibodies in the animal.

An additional embodiment of the invention comprises binding proteinswhich specifically bind to a peptide selected from the group consistingof: KSSGKLISL (SEQ ID NO:4), CNGRLYCGP (SEQ ID NO:5) and GTKLVCFAA (SEQID NO:6). A preferred embodiment comprises a binding protein wherein theprotein is an antibody.

An alternative embodiment of the invention comprises an antibody whichspecifically binds to a peptide sequence selected from the groupconsisting of: KSSGKLISL (SEQ ID NO:4), CNGRLYCGP (SEQ ID NO:5) andGTKLVCFAA (SEQ ID NO:6). Variants of antibodies (including an antigenbinding site), such as chimeric antibodies, humanized antibodies,veneered antibodies, and recombinantly engineered single chainantibodies which bind to the peptides of the present invention areincluded within the scope of the invention.

The invention additionally includes a method for inducing passiveimmunity in a host against HIV-1 comprising the step of administering anamount of antibody which specifically binds to a protein selected fromthe group consisting of: KSSGKLISL (SEQ ID NO:4), CNGRLYCGP (SEQ IDNO:5) and GTKLVCFAA (SEQ ID NO:6) said amount sufficient to inducepassive immunity against HIV-1.

An alternative embodiment comprises a method for detecting HIV-1 inbiological samples said method comprising detecting the presence ofHIV-1 in a sample with an antibody which specifically binds to a proteinselected from the group consisting of: KSSGKLISL (SEQ ID NO:4),CNGRLYCGP (SEQ ID NO:5) and GTKLVCFAA (SEQ ID NO:6) in an amountsufficient to detect the presence of HIV-1 in a sample.

A further embodiment of the invention comprises a method for detectingHIV-specific antibodies in a person suspected of being infected withHIV-1 said method comprising the step of incubating a biological samplefrom the person with an antigenic determinant selected from the groupconsisting of KSSGKLISL (SEQ ID NO:4), CNGRLYCGP (SEQ ID NO:5) andGTKLVCFAA (SEQ ID NO:6) in an amount sufficient to detect the presenceof antibodies which bind to the antigenic determinant.

Other embodiments of the invention include a method for selecting forantibodies specific to patients with long term nonprogression (LTNP)into AIDS said method comprising: (a) screening serum from LTNP patientsfor HIV specific antibodies and comparing the antibodies to patientswith AIDS.

Further, the invention includes peptides specific to antibodies frompatients with long term nonprogression (LTWP) into AIDS said peptidesgenerated via a method comprising: (a) screening serum from LTNPpatients for HIV-specific antibodies, and; (b) comparing the antibodiesto patients with AIDS.

An alternative embodiment comprises phagotopes having peptidesexhibiting antigens specific to antibodies found in patients with longterm nonprogression (LTNP) into AIDS.

Also included in the invention is an embodiment comprising phagotopeshaving peptides exhibiting antigens specific to antibodies found inpatients with long term nonprogression (LTNP) into AIDS said phagotopesproduced by screening serum from LTNP patients for HIV-specificantibodies against a random library of phagotopes, and; (b) comparingthe antibodies to patients with AIDS.

A further embodiment of the invention comprises an antigenic peptide ofless than 100 amino acids having an antigenic subsequence selected fromthe group consisting of EATVVYPAP (SEQ ID NO:7), TKTLIYGGA (SEQ IDNO:8), KRIVIGPQT (SEQ ID NO:9), CCGCLTCSV (SEQ ID NO:10), SGRLYCHESW(SEQ ID NO:11), FALSHYDKP (SEQ ID NO:12), and RPTLRFQGA (SEQ ID NO:13).

Embodiments of the invention also include a vaccine for protectingagainst HIV-1 infection comprising an antigenic peptide of less than 100amino acids having an antigenic subsequence selected from the groupconsisting of EATVVYPAP (SEQ ID NO:7), TKTLIYGGA (SEQ ID NO:8),KRIVIGPQT (SEQ ID NO:9), CCGCLTCSV (SEQ ID NO: 10), SGRLYCHESW (SEQ IDNO: 11), FALSHYDKP (SEQ ID NO: 12), and RPTLRFQGA (SEQ ID NO:13).

An additional embodiment of the invention comprises an antigenic peptideof less than 100 amino acids having an antigenic subsequence selectedfrom the group consisting of EGEFCKSSGKLISLCGDPAK (SEQ ID NO: 14),EGEFCQTKLVCFAAAGDPAK (SEQ ID NO:15), EGEFCCNGRLYCQPCGDPAK (SEQ ID NO:16), EGEFCCAGQLTCSVCGDPAK (SEQ ID NO: 17), CSGRLYCHESWC (SEQ ID NO: 18),and TKTLIYQGA (SEQ ID NO: 19).

The invention also includes a vaccine for protecting against HIV-1infection comprising an antigenic peptid of less than 100 amino acidshaving an antigenic subsequence selected from the group consisting ofEGEFCKSSGKLISLCGDPAK (SEQ ID NO: 14), EGEFCQTKLVCFAAAGDPAK (SEQ IDNO:15), EGEFCCNGRLYCQPCGDPAK (SEQ ID NO:16), EGEFCCAGQLTCSVCGDPAK (SEQID NO:17), CSGRLYCHESWC (SEQ ID NO:18), and TKTLIYQGA (SEQ ID NO:19).

A further diagnostic embodiment includes the use of peptides of theinvention to determine prognosis or disease progression in persons withchronic diseases or infections.

As will be apparent from the discussion below, other methods andembodiments are also contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. ELISA reactivities of HIV-specific phagotopes. a, LTNP and AIDSsubjects were selected as reported (Pantaleo et al., N. Engl. J Med332:209 (1995)). HIV-specific phagotopes were identified from RPLpVIII9aa-cys (selection 1), or pVIII9aa (selection 2) by serial steps ofscreening and phage colony purification as described in the Methodssection. Results are expressed as fold increase of the average values ofthe phagotopes over values of the wild-type phages. Values wereconsidered positive when at least four-fold higher than the backgroundsignal of wild-type phage. Average values of at least four independentassays are shown as <4; 4<<10; 10.1<<20; >20. The far right columnindicates the recognition frequency of positive sera by each phagotope(f). The percentages of significant phage reactivity in the HIV-positiveand control groups were compared using the Fisher Exact Test; p values(p<0.001) were adjusted for multiple testing using the Bonferronimethod. b, Comparison of ELISA reactivities of phagotopes with sera ofLTNP (hatched bars) versus AIDS subjects (black bars). Data areexpressed as mean±SEM of fold increase for each phagotope. Statisticalanalysis was performed according to the one-sided Student's t test;asterisks indicate p<0.05.

FIG. 2. Amino acid sequences of the HIV-specific phagotopes. The aminoacid sequences of peptides displayed on the HIV-specific phagotopes areshown as single letter codes. a, Homology between the amino acidsequences of p195, p217 and p197 and discrete regions of HIV gp160. Grayboxes indicate identity; similarity among amino acid residues isindicated as gray shading. b, consensus homology of p197 with a gp41domain conserved between HIV-1 subtypes A through G. c, Amino acidsequences of epitopes with no obvious sequence homology with HIV proteindomains.

FIG. 3. Phagotope-specific antibodies bind to HIV-1. a, ELISAreactivities of immunoaffinity purified antibodies with HIV-1.Antibodies were immunoaffinity purified from LTNP serum 6090 usingsingle phagotopes as ligands and tested for ELISA reactivity againstHIV-1 virions by using a standard ELISA kit (Organon). Purifiedantibodies were tested at 5-10 ng/ml; HIV-negative (CS) and serum 6090(HIV-1S) were tested at 1:100 dilution. Data are expressed as mean±SEMof four independent determinations. b, The binding of phagotope-specificantibodies to HIV-1 is specifically displaced by the related phagotopes.ELISA reactivities of single immunopurified antibodies to HIV-I weretested in the presence of the indicated concentrations of p195 (▪), p197(♦), p217 (●), p287 (▴), p335(▾). The binding of each antibody to HIVwas also tested in presence of increasing concentrations of wild-typephages (open symbols). c, Displacement of HIV-1 binding by peptidescorresponding to the phage-displayed epitopes shown in FIG. 2 a,c. ELISAreactivities of single immunoaffinity purified antibodies with therelated phagotopes were tested in the presence of increasingconcentrations of peptides 195 (▪), 197 (▴), 217 (♦), 287 (ρ), 335 (●).d, HIV-1 immunoblotting with phagotope-specific human antibodies.Immunoffinity purified antibodies were tested at 60 ng/ml for binding toHIV-1 proteins in Western blot by a diagnostic kit (Cambridge Biotech.);6090 LTNP serum was tested at 1:1000 dilution.

FIG. 4. ELISA reactivities of monkey sera with HIV-specific phagotopes.Sera of naive monkeys (SHIV Negative) and SHIV-infected animals weretested for binding to HIV-1 phagotopes. Results are expressed as foldincrease of OD_(405 nm) values of tested phagotope over the OD_(405 nm)values of wild-type-phage. Cutoff values were set as detailed in thelegend to FIG. 1 a. All the pre-infection sera of SHIV-positive animalstested negative by ELISA (not shown). A125, 42C, E50 and AK98 are Rhesusmacaques; 4138, 4150 and 79 are cynomologous macaques; these animalswere infected with SHIV_(MD1) (Shibata et al., J. Infect. Dis. 176:362(1997)). 17860 and 17846 are pigtail macaques infected withSHIV_(MD14YE) (Shibata et al., J. Infect. Dis. 176:362 (1997)).

FIG. 5. Assays of the neutralization of HIV-1 by phagotope-specificantibodies. C57B16 mice were immunized with either wild-type phages (ρ)or with p195 (▪), p197 (□), p217 ( ), p287 ( ) ), p335 (▴)). IgG werepurified from immunized mice and tested for inhibition of HIV_(IIIB) (a)or NL4-3 (b) infection in the MT2 assay (Montefiori et al., J Clin.Microbiol. 26:231 (1988)). Neutralization of AD8 infection was performedon PHA-activated PBMC (Montefiori el al, J. Virol. 72:1886 (1998)) (c).Results are expressed as percentages of protection and arerepresentative of three independent experiments. IgG from BALB/cimmunized mice gave comparable results (not shown).

FIG. 6. ELISA reactivities of sera from Rhesus macaque monkeysinoculated with (a) phage-displayed epitopes or (b) WT-phage.

FIG. 7. ELISA reactivity of sera from Rhesus macaque monkeys that wasassessed for binding to HIV-IIIB gp160.

DETAILED DESCRIPTION

Introduction

This invention provides for peptides and antibodies which are associatedwith long term survival of persons infected with HIV-1. Immunogenicpeptides are identified herein and are correlated with HIV-specificproteins. Other immunogenic peptides are conformational equivalents ofepitopes on HIV-1 proteins and have little amino acid identity to knownHIV-1 proteins. The following teaches those of skill how to make and usethe peptides of this invention.

Definitions

Binding Protein: A binding protein is a protein which binds specificallyto a target ligand. The term includes both antibodies and proteinsgenerated by random selection such as those displayed on phage.

Antibody: As used herein, an “antibody” refers to a protein functionallydefined as a binding protein and structurally defined as comprising anamino acid sequence that is recognized by one of skill as being derivedfrom the framework region of an immunoglobulin encoding gene of ananimal producing antibodies. An antibody can consist of one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

Antibodies exist as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, e.g., Fundamental Immunology, W. E. Paul, ed., RavenPress, N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Preferred antibodies include single chainantibodies (antibodies that exist as a single polypeptide chain), morepreferably single chain Fv antibodies (sFv or scFv) in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide. The singlechain Fv antibody is a covalently linked VH-VL heterodimer which may beexpressed from a nucleic acid including VH- and VL-encoding sequenceseither joined directly or joined by a peptide-encoding linker. Huston etal., Proc. Nat. Acad. Sci. USA 85:5879-5883 (1988). While the VH and VLare connected to each as a single polypeptide chain, the VH and VLdomains associate non-covalently. The first functional antibodymolecules to be expressed on the surface of filamentous phage weresingle-chain

Fv's (scFv), however, alternative expression strategies have also beensuccessful. For example Fab molecules can be displayed on phage if oneof the chains (heavy or light) is fused to g3 capsid protein and thecomplementary chain exported to the periplasm as a soluble molecule. Thetwo chains can be encoded on the same or on different replicons; theimportant point is that the two antibody chains in each Fab moleculeassemble post-translationally and the dimer is incorporated into thephage particle via linkage of one of the chains to g3p (see, e.g., U.S.Pat. No. 5,733,743). The scFv antibodies and a number of otherstructures converting the naturally aggregated, but chemically separatedlight and heavy polypeptide chains from an antibody V region into amolecule that folds into a three dimensional structure substantiallysimilar to the structure of an antigen-binding site are known to thoseof skill in the art (see, e.g., U.S. Pat. Nos. 5,091,513; 5,132,405; and4,956,778). Particularly preferred antibodies include all those thathave been displayed on phage (e.g., scFv, Fv, Fab and disulfide linkedFv (Reiter et al., Protein Eng., 8:1323-1331 (1995)). Antibodies canalso include diantibodies and miniantibodies.

Antibody Combining Site: An antibody combining site is that structuralportion of an antibody molecule comprised of a heavy and light chainvariable and hypervariable regions that specifically binds (immunoreactswith) an antigen. The term immunoreact in its various forms meansspecific binding between an antigenic determinant-containing moleculeand a molecule containing an antibody combining site such as a wholeantibody molecule or a portion thereof.

Antigenic: Antigenic refers to the ability of a composition to give riseto antibodies specific to itself or to give rise to a cell-mediatedimmune response.

Antigenic determinant: An antigenic determinant or epitope is the siteof recognition or binding of an antibody to its target or the site ofrecognition or binding of a T cell receptor. It is minimally defined by4-6 amino acids. It can be linear or conformational.

Phagotope: A peptide displayed on phage bound by serum antibodies.

Monoclonal Antibody: A monoclonal antibody in its various grammaticalforms refers to a population of antibody molecules that contain only onespecies of antibody combining site capable of immunoreacting with aparticular epitope. A monoclonal antibody thus typically displays asingle binding affinity for any epitope with which it immunoreacts. Amonoclonal antibody may therefore contain an antibody molecule having aplurality of antibody combining sites, each immunospecific for adifferent epitope, erg., a bispecific monoclonal antibody.

Fusion Polypeptide: A polypeptide comprised of at least two polypeptidesand a linking sequence to operatively link the two polypeptides into onecontinuous polypeptide. The two polypeptides linked in a fusionpolypeptide are typically derived from two independent sources, andtherefore a fusion polypeptide comprises two linked polypeptides notnormally found linked in nature.

Synthetic peptide: A peptide that is not naturally occurring, but isman-made using methods such as chemical synthesis or recombinant DNAtechnology.

Vaccines: Vaccines refer to compositions or mixtures that whenintroduced into the circulatory system of an animal will evoke aprotective response to a pathogen. Vaccines can be either passive oractive. Vaccines are passive when they include immunoglobulins whichconfer protection and are active when they elicit from the hostantibodies which are protective against a pathogen.

Method for identifying antibodies specific to persons resistant todisease progression.

Persons harboring infectious agents that are initially dormant and thenprogress into clinical disease states present a problem to clinicians.Persons who do not rapidly progress to the clinical state are consideredresistant. Often the reason for a person's resistance is unknown but theimmune system is thought to play a part in the delay of clinicalmanifestations of the disease. HIV-1 infection is a classic example ofthis phenomenon, but other diseases such as Herpes simplex and Herpeszoster infections, lyme disease, infection with the hepatitis viruses,and tuberculosis follow this general pattern.

To identify antibodies specific to resistance one simply collects arepresentative sample of the antibodies in healthy, chronically infectedand acutely infected persons. The antibodies are then screened andcomparisons made to determine the unique antibodies to the pathogensthat are present in the resistant persons. There are numerous means toachieve this which would be immediately apparent to those of skill oncethe importance of the selection of resistant persons is noted as it wasin the instant situation with HIV-1.

More particularly, one immobilizes the antibodies from infected personsand independently creates a random peptide library using any number ofmeans including synthetic peptide chemistry, phage display technology,and other virally based peptide display technology. Methods for theproduction of phage display libraries, including vectors and methods ofdiversifying the population of peptides which are expressed, are wellknown in the art. (See, for example, Smith and Scott, Methods Enzymol.217:228-257 (1993); Scott and Smith, Science 249:386-390 (1990); andHuse, WO 91/07141 and WO 91/07149. Cyclic peptide libraries also arewell known in the art (see, for example, Koivunen et al., MethodsEnzymol. 245:346-369 (1994)). These or other well known methods can beused to produce a phage display library

The library is blocked by contact with non-immobilized antibodies fromhealthy sera, or selectively screened for disease-specific peptidesusing an immunoaffinity column prepared with healthy sera and the eluantsubsequently used for further selection. In either case, thedisease-enriched library is then contacted with immobilized antibodiesfrom resistant persons or from acutely infected persons and the twopopulations of antibodies are contrasted using conventional technologyto identify the antibodies that are specifically found in the sera ofresistant individuals.

Alternatively, a strategy based on positive selection may be employedwherein a pool of clones bound by serum antibodies from a singleresistant (or acutely infected) person is identified using animmunoaffinity column. The pool of clones is then individually screenedfor those clones that react with a second, different serum from a personof the same disease status as that of the source of the first serum. Thepositive clones identified with the second serum are then analyzed fortheir frequency and selectivity of reaction with sera from resistant oracutely infected persons and healthy persons using conventionaltechniques such as ELISA.

In the example section, one procedure is provided; however, those ofskill should recognize that a number of variations on this procedurewill lead to the identification of antibodies and antigens that arespecific to those persons demonstrating long term resistance to adisease or infection.

Manufacture of Reptides for raising HIV-specific antibodies

The peptides of the invention can be prepared in a wide variety of ways.The peptides can be synthesized in solution or on a solid support inaccordance with conventional techniques. Various automatic synthesizersare commercially available and can be used in accordance with knownprotocols. See, for example, Stewart & Young, SOLID PHASE PEPTIDESYNTHESIS, 2D. ED., Pierce Chemical Co. (1984).

Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of interest is inserted intoan expression vector, transformed or transfected into an appropriatehost cell and cultivated under conditions suitable for expression. Itwill be understood by those of skill in the art that peptidecompositions containing multiple peptides may be produced by engineeringa nucleic acid sequence to encode a fusion protein comprising themultiple peptide sequences. These procedures are generally known in theart, as described generally in, for example, Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989).

Expression vectors suitable for use in the present invention maycomprise at least one expression control element operationally linked tothe nucleic acid sequence. Expression control elements are inserted inthe vector to control and regulate the expression of the nucleic acidsequence. Additional preferred or required operational elements include,but are not limited to, a leader sequence, termination codons,polyadenylation signals, and any other sequences necessary or preferredfor the appropriate transcription and subsequent translation of thenucleic acid sequence in the host system. It will be understood by oneskilled in the art the correct combination of required or preferredexpression control elements will depend on the host system chosen. Itwill further be understood that the expression vector should containadditional elements necessary for the transfer and subsequentreplication of the expression vector containing the nucleic acidsequence in the host system. Examples of such elements include, but arenot limited to, origins of replication and selectable markers. It willfurther be understood by one skilled in the art that such vectors areeasily constructed using conventional methods (see, e.g., Ausubel et al.in “Current Protocols in Molecular Biology”, John Wiley and Sons, NewYork, N.Y. (1987)) or are commercially available.

Modification of Peptides

With regard to protein based vaccines of the invention, there are anumber of strategies for amplifying an immunogen's effectiveness,particularly as related to the art of vaccines. These include strategieswhereby an immunogenic peptide may be directly modified to enhanceimmunogenicity or physical properties such as stability. For example,cyclization or circularization of a peptide can increase the peptide'santigenic and immunogenic potency. See, e.g., U.S. Pat. No. 5,001,049which is incorporated by reference herein.

The immunogenicity of the peptides of the present invention may also bemodulated by coupling to fatty acid moieties to produce lipidatedpeptides. Convenient fatty acid moieties include glycolipid analogs,N-palmityl-S2RS)2,3-bis-(palmitoyloxy)pmopyl-cysteinyl-serine (PAM3Cys-Ser), N-palmityl-S-[2,3 bis (palmitoyloxy)-(2RS)-propyl-[R]-cysteine(TPC) or adipalmityl-lysine moiety

The peptides may also be conjugated to a lipidated amino acid, such asan octadecyl ester of an aromatic acid, such as tyrosine, includingactadecyl-tryrosine (OTH).

Protein Analogs

Protein analogs are defined functionally as those compounds that willact chemically and biologically as the peptides provided herein. Inparticular the invention includes analogs which bind with fidelity tothe antibodies which are generated using the peptides described herein.The analogs will find advantage as more stable and as having longer halflife under in vivo conditions. In addition, it may also be advantageousto modify the peptides in order to impose a conformational restraintupon it. This may be useful, for example, to mimic a naturally-occurringconformation of the peptide in the context of the native protein inorder to optimize the effector immune responses that are elicited.Modified peptides are referred to herein as “analog” peptides. The term“analog” extends to any functional and/or structural equivalent of apeptide characterized by its increased stability and/or efficacy in vivoor in vitro in respect of the practice of the invention. The term“analog” also is used herein to extend to any amino acid derivative ofthe peptides as described herein.

Analogs of the peptides contemplated herein include, but are not limitedto, modifications to side chains; and incorporation of unnatural aminoacids and/or their derivatives, non-amino acid monomers andcross-linkers. Other methods which impose conformational constraint onthe peptides or their analogs are also contemplated.

It will be apparent that the peptide of the invention can be modified ina variety of different ways without significantly affecting thefunctionally important immunogenic behavior thereof. Possiblemodifications to the peptide sequence may include the following:

One or more individual amino acids can be substituted by amino acidshaving comparable or similar properties, thus, for example,:

-   V may be substituted by I;-   T may be substituted by S;-   K may be substituted by R; and-   L may be substituted by I, V or M.

One or more of the amino acids of the peptides of the invention can bereplaced by a “retro-inverso” amino acid, i.e., a bifunctional aminehaving a functional group corresponding to an amino acid, as discussedin WO 91/13909

One or more amino acids can be deleted or added. Added amino acids may,for example, comprise residues that correspond to phage coat proteinsequences that are adjacent to the phagotope sequence.

Structural analogs mimicking the 3-dimensional structure of the peptidecan be used in place of the peptide itself.

Examples of side chain modifications contemplated by the presentinvention include modification of amino groups, such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzenesulfphonic acid (TNBS); acylation of amino groups with succinicanhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysinewith pyridoxal-5′-phosphate followed by reduction with NaBH₄.

The guanidino group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivatization, forexample, to a corresponding amide.

Sulfhydryl groups may be modified by methods such as carboxy-methylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of mixed disulphides with other thiol compounds;reaction with maleimide, maleic anhydride or other substitutedmaleimide; formation of mercurial derivatives using4-chloromercur-ibenzoate, 4-chloromercuriphenylsulfonic acid,phenylmercury chloride, 2-chloromercuric-4-nitrophenol and othermercurials; and carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tryosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butyglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienylalanine, and/or D-isomers of amino acids.

Crosslinkers can be used, for example, to stabilize 3-dimensionalconformations, using homo-bifunctional crosslinkers such as thebifunctional imido esters having (CH2)[n], spacer groups with n=1 ton=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctionalreagents which usually contain an amino-reactive moiety such asN-hydroxysuccinimide and another group specific-reactive moiety such asmaleimido or dithio (for SH) or carbodiimide (for COOH). In addition,peptides could be conformationally constrained by, for example,incorporation of alpha-methylamino acids, introduction of double bondsbetween adjacent C atoms of amino acids and the formation of cyclicpeptides or analogs by introducing covalent bonds such as forming anamide bond between N and C termini, between two side chains or between aside chain and the N or C terminus.

Conjugation to other peptides or polypeptides

The peptides of the invention or their analogs may occur as a singlelength or as multiple tandem or non-tandem repeats. A single type ofpeptide or analog may form the repeats or the repeats may be composed ofdifferent molecules including a suitable carrier.

The use of the peptides provided herein under in viva conditions mayrequire their chemical modification since the peptides themselves maynot have a sufficiently long scrum and/or tissue half-life. For thispurpose, the peptides may optionally be linked to a carrier molecule,possibly via chemical groups of amino acids of the peptide or viaadditional amino acids added at the C- or N-terminus.

A small peptide antigen can be conjugated to a suitable carrier, usuallya protein molecule, to enhance its immunogenicity. This procedure hasseveral facets. It can allow multiple copies of an antigen, such as apeptide, to be conjugated to a single larger carrier molecule.Additionally, the carrier may possess properties which facilitatetransport, binding, absorption or transfer of the antigen. Theconjugation between a peptide and a carrier can be accomplished usingone of the methods known in the art. Specifically, conjugation can beperformed using bifunctional cross-linkers as binding agents asdetailed, for example, by Means and Feeney, Bioconjugate Chem. 1:2-12(1990). Many suitable linkages are known, e.g., using the side chains ofTyr residues. Suitable carriers are well known in the art, and include,e.g., keyhole limpet hemocyanin (KLH), thyroglobulin, serum albumin,purified protein derivative of tuberculin (PPD), ovalbumin, tetanustoxoid, non-protein carriers and many others.

The immunogenicity of the peptide compositions of the present inventionmay further be enhanced by linking the peptides to one or more peptidesequences that are able to a elicit a cellular immune response (see,e.g., WO94/20127). Peptides that stimulate cytotoxic T-lymphocyte (CTL)responses as well as peptides that stimulate helper T lymphocyte (HTL)responses are useful for linkage to the peptides of the invention. Thepeptides may be linked by a spacer molecule. The spacer is typicallycomprised of relatively small, neutral molecules, such as amino acids oramino acids mimetics, which are uncharged under physiologicalconditions.

A peptide of the invention may be linked to a T helper peptide that isrecognized by T helper cells in the majority of the population. This canbe accomplished by selecting amino acid sequences that bind to many,most, or all of the HLA class II molecules. An example of such a Thelper peptide is tetanus toxoid at positions 830-843 (see, e.g.,Panina-Bordignon et al., Eur. J. Immunol., 19:2237-2242 (1989)).

Further, a peptide may be linked to multiple antigenic determinants toenhance immunogenicity. For example, in order to elicit recognition by Tcells of multiple HLA types, a synthetic peptide encoding multipleoverlapping T cell antigenic determinants (cluster peptides) may be usedto enhance immunogenicity (see, e.g., Ahlers et al., J. Immunol.150:5647-5665 (1993). Such cluster peptides contain overlapping, butdistinct antigenic determinants. The cluster peptide may be synthesizedcolinearly with a peptide of the invention. In one embodiment, thecluster peptide may be positioned at the amino terminal end of a peptideof the invention. The cluster peptide may be linked to a peptide of theinvention by one or more spacer molecules.

A peptide composition comprising a peptide of the invention linked to acluster peptide may also be used in conjunction with a cluster peptidelinked to a CTL-inducting epitope. Such compositions may be administeredvia alternate routes or using different adjuvants.

Alternatively multiple peptides encoding CTL and/or HTL epitopes may beused in conjunction with a peptide of the invention.

One embodiment for the use of multiple peptide epitopes known as themultiple antigen peptide system (MAP), utilizes a small peptidyl corematrix with covalently attached, radially branching, multiple syntheticpeptides. See, for example, Tam, J. P., Proc. Natl. Acad. Sci. U.S.A.85:5409-5413 (1988). The MAP system is a combination antigen/antigencarrier that is composed of two or more antigenic molecules covalentlyattached to a dendritic core that is comprised of bifunctional units.The dendritic core of a multiple antigen peptide system can be composedof lysine molecules. For example, a lysine is attached via peptide bondsthrough each of its amino groups to two additional lysines. This secondgeneration molecule has four free amino groups each of which can becovalently linked to an additional lysine to form a third generationmolecule with eight free amino groups. A peptide may be attached to eachof these free groups to form an octavalent multiple peptide antigen.Alternatively, the second generation molecule having four free aminogroups can be used to form a tetravalent MAPS, i.e., a MAPS having fourpeptides covalently linked to the core. Many other molecules, includingaspartic acid and glutamic acid, can be used to form the dendritic coreof a multiple peptide antigen system. The dendritic core, and the entireMAPS may be conveniently synthesized on a solid resin using the classicMerrifield synthesis procedure.

Multiple antigen peptide systems have many advantages as antigen carriersystems. Their exact structure and composition is known; the ratio ofantigen to carrier is quite high; and several different antigens, e.g.,a B cell epitope such as a peptide of the invention, and a T cellepitope, may be attached to a single dendritic core. When both a B cellepitope and a T cell epitope are present it is preferable that they arelinked in tandem on the same functional group of the dendritic core.Alternatively, the T cell epitope and the B cell epitope may be onseparate branches of the dendritic core. The T-cell epitope may be a CTLor HTL-inducing antigenic determinant.

Pseudomonas toxin conjugation

In another embodiment, the peptides of this invention provides a vaccinecomprising a chimeric Pseudomonas exotoxin A (PE) protein in which thepeptides of this invention are inserted into the Ib domain of theexotoxin. Such chimeric constructs are described in FitzGerald et al.,J. Biol. Chem., 273:9951 (1998).

Pseudomonas exotoxin A has been shown to act as a carrier-adjuvant forantigens. The protein comprises three prominent globular domains (Ia, IIand III) and one small subdomain (Ib). Domain Ia binds to a receptor onmost mammalian cell surfaces. Domain II translocates the protein intothe cytosol. Domain II has ADP-ribosylating activity which shuts downprotein synthesis. The protein can be made non-toxic by, for example,deleting amino acid E553. The protein also can be directed to differentcells by exchanging the cell binding domain with ligands for otherreceptors or antibodies. It comprises a loop formed from a disulfidebond between two amino acids in the domain.

Various genetically modified forms of PE are described, e.g., in U.S.Pat. Nos. 5,602,095; 5,512,658; 5,458,878, and in FitzGerald et al.,PCT/US98/14341.

FitzGerald et al. teach a method for replacing amino acid sequences inthis loop with sequences from HIV which is applicable to the peptides ofthis invention. They showed that the non-toxic form of this chimericprotein could elicit HIV-neutralizing antibodies when injected intorabbits. Furthermore, because the chimera gains entry into the cytosol,it may result in the generation of viral peptides and presentation viamajor histocompatibility complex class I antigens.

Accordingly, this invention provides a recombinant nucleic acid thatcomprises a nucleotide sequence encoding a chimeric Pseudomonas exotoxinA protein wherein a nucleotide sequence encoding a peptide of thisinvention is inserted into a nucleotide sequence encoding the lb loop ofPseudomonas exotoxin A. The peptide of this invention can merely beinserted into the loop or can replace part or all of the loop. In analternative embodiment, the nucleotide sequence can comprise anucleotide sequence encoding a ligand for a receptor of choice, whereinthe ligand replaces all or part of the Ia domain. In another embodiment,the recombinant nucleic acid is an expression vector comprising anexpression control sequence operatively linked to the nucleotidesequence encoding the chimeric immunogen. A host cell can be transfectedwith the recombinant nucleic acid and the chimeric protein can beexpressed thereby.

FitzGerald et al. showed that the chimeric protein can be expressed in abacterial cell and properly folded so as to have activity. The resultingchimeric immunogens can be used in a vaccine to immunize persons againstHIV.

Preparation of peptide-specific antibodies

Monoclonal Antibodies

The monoclonal antibodies of the invention can be made by conventionaltechniques which are commonly used in hybridoma production (see, e.g.CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY; and Antibodies ALaboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press,1989). In brief, mice are immunized with the peptides of this invention.B-cells are taken from the spleens of the immunized mice and fused withNS-1 myeloma cells. Polyethylene glycol mixed with dimethyl suffoxide(DMSO) in calcium- and magnesium-free phosphate buffered saline (PBS)can be used as the fusion reagent. The hybridomas generated from thefusion are then transferred to 96 well microtiter plates and grown.

Polyclonal Antibodies

Methods of production of polyclonal antibodies are known to those ofskill in the art (e.g., Wiley/Greene and Harlow & Lane, ibid.). Inbrief, an immunogen, preferably a purified protein, is mixed with anadjuvant and animals are immunized. The animal's immune response to theimmunogen preparation is monitored by taking test bleeds and determiningthe titer of reactivity to the administered protein. When appropriatelyhigh titers of antibody to the immunogen are obtained, blood iscollected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be performed if desired.

Single Chain Antibodies

Preferred antibodies include single chain antibodies (antibodies thatexist as a single polypeptide chain), more preferably single chain Fvantibodies (sFv or scFv) in which a variable heavy and a variable lightchain are joined together (directly or through a peptide linker) to forma continuous polypeptide. The single chain Fv antibody is a covalentlylinked VH-VL heterodimer which may be expressed from a nucleic acidincluding VH- and VL-encoding sequences either joined directly or joinedby a peptide-encoding linker. Huston et al., Proc. Nat. Acad. Sci. USA,85:5879-5883 (1988). While the VH and VL are connected to each as asingle polypeptide chain, the VH and VL domains associatenon-covalently. The first functional antibody molecules to be expressedon the surface of filamentous phage were single-chain Fv's (scFv),however, alternative expression strategies have also been successful.For example Fab molecules can be displayed on phage if one of the chains(heavy or light) is fused to g3 capsid protein and the complementarychain exported to the periplasm as a soluble molecule. The two chainscan be encoded on the same or on different replicons; the importantpoint is that the two antibody chains in each Fab molecule assemblepost-translationally and the dimer is incorporated into the phageparticle via linkage of one of the chains to g3p (see, e.g., U.S. Pat.No. 5,733,743). The scFv antibodies and a number of other structuresconverting the naturally aggregated, but chemically separated light andheavy polypeptide chains from an antibody V region into a molecule thatfolds into a three dimensional structure substantially similar to thestructure of an antigen-binding site are known to those of skill in theart (see, e.g., U.S. Pat. Nos. 5,091,513; 5,132,405; and 4,956,778).Particularly preferred antibodies include all those that have beendisplayed on phage (e.g., scFv, Fv, Fab and disulfide linked Fv (Reiteret al., Protein Eng,. 8:1323-1331 (1995)). Antibodies can also includediantibodies and miniantibodies.

Formulation of Immunogenic Compositions

Immunogenic compositions suitable for use as vaccines may be preparedfrom immunogenic peptides as disclosed herein. The immunogeniccomposition elicits an immune response which produces antibodies thatare opsonizing or antiviral. Should the vaccinated subject be challengedby HIV-1, the antibodies bind to the virus and thereby inactivate it.

Vaccines containing peptides are generally well known in the art, asexemplified by U.S. Pat. Nos. 4,601,903; 4,599,231; 4,599,230; and4,596,792. Vaccines may be prepared as injectables, as liquid solutionsor emulsions. The peptides may be mixed with pharmaceutically-acceptableexcipients which are compatible with the peptides. Excipients mayinclude water, saline, dextrose, glycerol, ethanol, and combinationsthereof. The vaccine may further contain auxiliary substances such aswetting or emulsifying agents, pH buffering agents, or adjuvants toenhance the effectiveness of the vaccines.

More specifically, the immunogens of this invention may be combined ormixed with various solutions and other compounds as is known in the art.For example, an immunogen may be administered in water, saline orbuffered vehicles with or without various adjuvants or immunodilutingagents. Examples of such adjuvants or agents include aluminum hydroxide,aluminum phosphate, aluminum potassium sulfate (alum), berylliumsulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-wateremulsions, muramyl dipeptide, bacterial endotoxin, lipid X,Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis,polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A,saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers orother synthetic adjuvants. Such adjuvants are available commerciallyfrom various sources, for example, Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and CompleteAdjuvant (Difco Laboratories, Detroit, Mich.). Other suitable adjuvantsare Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or amixture of Amphigen and Alhydrogel. Only aluminum is approved for humanuse.

Conveniently, the vaccines are formulated to contain a finalconcentration of immunogen in the range of from 0.2 to 200 μg/ml,preferably 5 to 50 μg/ml, most preferably 15 μg/ml. After formulation,the vaccine may be incorporated into a sterile container which is thensealed and stored at a low temperature, for example 4° C., or it may befreeze-dried. Lyophilization permits long-term storage in a stabilizedform.

For suppositories, binders and carriers may include, for example,polyalkalene glycols or triglycerides. Oral formulations may includenormally employed incipients such as, for example, pharmaceutical gradesof saccharine, cellulose and magnesium carbonate. These compositionstake the form of solutions, suspensions, tablets, pills, capsules,sustained release formulations or powders and contain 10-95% of thepeptides.

The peptides of this invention can be formulated for administration viathe nasal passages. Formulations suitable for nasal administration,wherein the carrier is a solid, include a coarse powder having aparticle size, for example, in the range of about 10 to about 500microns, which is administered in the manner in which snuff is taken,ie., by rapid inhalation through the nasal passage from a container ofthe powder held close up to the nose. Suitable formulations wherein thecarrier is a liquid for administration as, for example, nasal spray,nasal drops, or by aerosol administration by nebulizer, include aqueousor oily solutions of the active ingredient. For further discussions ofnasal administration of AIDS-related vaccines, references are made tothe following U.S. Pat. Nos.: 5,846,978; 5,663,169; 5,578,597;5,502,060; 5,476,874; 5,413,999; 5,308,854; 5,192,668; and 5,187,074.

Administration

The vaccines may be administered by any conventional methods includingoral administration and parenteral (e.g., subcutaneous or intramuscular)injection. The treatment may consist of a single dose of vaccine or aplurality of doses over a period of time. The immunogen of the inventioncan be combined with appropriate doses of compounds including otherepitopes of the target bacteria. Also, the immunogen could be acomponent of a recombinant vaccine which could be adaptable for oraladministration.

The proportion of immunogen and adjuvant can be varied over a broadrange so long as both are present in effective amounts. For example,aluminum hydroxide can be present in an amount of about 0.5% of thevaccine mixture (Al₂O₃ basis). On a per-dose basis, the amount of theimmunogen can range from about 5 μg to about 100 μg protein per patientof about 70 kg. A preferable range is from about 20 μg to about 40 μgper dose. A suitable dose size is about 0.5 ml. Accordingly, a dose forintramuscular injection, for example, would comprise 0.5 ml containing20 μg of immunogen in admixture with 0.5% aluminum hydroxide.

Vaccines of the invention may be combined with other vaccines for otherdiseases to produce multivalent vaccines. A pharmaceutically effectiveamount of the immunogen can be employed with a pharmaceuticallyacceptable carrier such as a protein or diluent useful for thevaccination of mammals, particularly humans. Other vaccines may beprepared according to methods well-known to those skilled in the art.

The therapeutic application of AIDS vaccines can be done by way of nasaladministration. Various ways of such administration are known in theart. The pharmaceutical formulation for nasal administration may beprepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art. The unit dosage for nasal administration can be from 1 to 3000mg, preferably 70 to 1000 mg, and most preferably, 1 to 10 mg of activeingredient per unit dosage form.

Alternatively, other modes of administration including suppositories andoral formulations may be desirable.

The peptides of the present invention may also be administered inconjunction with immune stimulating complexes (ISCOMS) ISCOMS arenegatively charged cage-like structure of 30-40 nm in size formedspontaneously on mixing cholesterol and Quil A (saponin). Protectiveimmunity has been generated in a variety of experimental models ofinfection including toxoplasmosis and Epstein-Barr virus-induced tumorsusing ISCOMS as the delivery vehicle for antigens (see, e.g., Mowat andDonachie, Immunol. Today, 23:383-385 (1991)). Immunogenic compositionsusing ISCOMS are comprised of the peptides of the invention encapsulatedinto ISCOMS for delivery.

Immunotherapy regimens which produce maximal immune responses followingthe administration of the fewest number of doses, ideally only one dose,are highly desirable. This result can be approached through entrapmentof immunogen in microparticles. For example, the absorbable suturematerial poly(lactide-co-glycolide) co-polymer can be fashioned intomicroparticles containing immunogen (see, e.g., Eldridge et al., Molec.Immunol., 28:287-294 (1991); Moore et al., Vaccine 13:1741-1749 (1995);and Men et al., Vaccine, 13:683-689 (1995)). Following oral orparenteral administration, microparticle hydrolysis in vivo produces thenon-toxic byproducts, lactic and glycolic acids, and releases immunogenlargely unaltered by the entrapment process. Microparticle formulationscan also provide primary and subsequent booster immunizations in asingle administration by mixing immunogen entrapped microparticles withdifferent release rates. Single dose formulations capable of releasingantigen ranging from less than one week to greater than six months canbe readily achieved.

The peptides of the invention may also be administered via liposomes,which serve to target the peptides to a particular tissue, such aslymphoid tissue, or targeted selectively to infected cells, as well asincrease the half-life of the peptide composition. Liposomes includeemulsions, foams, micelles, insoluble monolayers, liquid crystals,phospholipid dispersions, lamellar layers and the like. In thesepreparations the peptide to be delivered is incorporated as part of aliposome, alone or in conjunction with a molecule which binds to, e.g.,a receptor prevalent among lymphoid cells, such as monoclonal antibodieswhich bind to the CD45 antigen, or with other therapeutic or immunogeniccompositions. Thus, liposomes either filled or decorated with a desiredpeptide of the invention can be directed to the site of lymphoid cells,where the liposomes then deliver the peptide compositions. Liposomes foruse in the invention are formed from standard vesicle-forming lipids,which generally include neutral and negatively charged phospholipids anda sterol, such as cholesterol. The selection of lipids is generallyguided by consideration of, e.g., liposome size, acid lability andstability of the liposomes in the blood stream. A variety of methods areavailable for preparing liposomes, as described in, e.g., Szoka, et al.,Ann. Rev. Biophys. Bioeng., 9:467 (1980), and U.S. Pat. Nos. 4,235,871;4,501,728; 4,837,028; and 5,019,369.

Nucleic acid vaccines

Nucleic acids (typically DNA) encoding the polypeptides of the inventionare administered to patients to elicit an immune response against thepolypeptides which they encode. DNA administered for this purpose isreferred to as a “DNA vaccine.” Methods of making and administering DNAas vaccines are known, and described, e.g., in Wolff et. al., Science,247:1465-1468 (1990).

In general, the dose of a naked nucleic acid composition such as a DNAvaccine or gene therapy vector is from about 1 μg to 100 μg for atypical 70 kilogram patient. The immunogenic composition can be either anucleic acid encoding the target protein (e.g., a DNA vaccine) or avirus vector which produces the antigenic protein. Subcutaneous orintramuscular doses for naked nucleic acid (typically DNA encoding afusion protein) will range from 0.1 μg to 500 μg for a 70 kg patient ingenerally good health. Subcutaneous or intramuscular doses for viralvectors comprising the fusion proteins of the invention will range from1×10⁵ pfu to 1×10⁹ for a 70 kg patient in generally good health.

Passive immunization

Passive immunotherapeutic methods are applicable to persons exhibitingsymptoms of HIV-induced disease, including AIDS or related conditionsbelieved to be caused by HIV infection, and humans at risk of HIVinfection. Patients at risk of infection by HIV include babies ofHIV-infected pregnant mothers, recipients of transfusions known tocontain HIV, users of HIV contaminated needles, individuals who haveparticipated in high risk sexual activities with known HIV-infectedindividuals, and the like risk situations.

HIV has been disclosed as treatable using passive immunization. See forexample Jackson et al., Lancet, September 17:647-652, (1988); Karpas etal., Proc. Natl. Acad. Sci, USA 87:7613-7616 (1990), Eichberg et al.,AIDS Res. Hum. Retroviruses 8:1515 (1992) and U.S. Pat. No. 5,830,476.Passive immunization can be accomplished with polyclonal antibodies,monoclonal antibodies, or antibody fragments.

In one embodiment, the passive immunization method comprisesadministering a composition comprising more than one species of humanmonoclonal antibody of this invention, preferably directed tonon-competing epitopes or directed to distinct serotypes or strains ofHIV, as to afford increased effectiveness of the passive immunotherapy.

A therapeutically (immunotherapeutically) effective amount of ahumanized or human antibody is a predetermined amount calculated toachieve the desired effect, i.e., to neutralize the HIV present in thesample or in the patient, and thereby decrease the amount of detectableHIV in the sample or patient. In the case of in vivo therapies ofpersons already infected, an effective amount can be measured byimprovements in one or more symptoms associated with HTV-induced diseaseoccurring in the patient, or by serological decreases in HIV antigens.

Thus, the relevant dosage ranges for the administration of themonoclonal or other antibodies of the invention are those large enoughto produce the desired effect in which the symptoms of the HIV diseaseare ameliorated or the likelihood of infection decreased. The dosageshould not be so large as to cause adverse side effects, such ashyperviscosity syndromes, pulmonary edema, congestive heart failure, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of the disease in the patient and can be determined by one ofskill in the art. The dosage can be adjusted by the individual physicianin the event of any complication.

A therapeutically effective amount of an antibody of this invention istypically an amount of antibody such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.1 μg/ml to about 100 μg/ml, preferablyfrom about 1 μg/ml to about 5 μg/ml, and usually about 5 μg/ml. Stateddifferently, the dosage can vary from about 0.1 mg/kg to about 300mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg, mostpreferably from about 0.5 mg/kg to about 20 mg/kg, in one or more doseadministrations daily, for one or several days.

The antibodies of the invention can be administered parenterally byinjection or by gradual infusion over time. Although the HIV infectionis typically systemic and therefore most often treated by intravenousadministration of therapeutic compositions, other tissues and deliverymeans are contemplated where there is a likelihood that the tissuetargeted contains infectious HIV. Thus, antibodies of the invention canbe administered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, transdermally, and can be delivered byperistaltic means.

The therapeutic compositions containing antibodies of this invention areconventionally administered intravenously, as by injection of a unitdose, for example. The term “unit dose” when used in reference to atherapeutic composition of the present invention refers to physicallydiscrete units suitable as unitary dosage for the subject, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requireddiluent; ie., carrier, or vehicle.

Diagonostic uses in immunoassays and other uses

Immunoassays

The peptides of this invention or or antibodies specific to the peptidesthemselves can be used to detect the presence of HIV in serum or in anybiological sample. The assays will find use in both medical and researchsettings.

Normally, the peptides are in the range of about 9 residues and up toabout 40 residues. The preferred range is 9 to 25 residues. There may becircumstances where a mixture of peptides from conserved regions and/orfrom the non-conserved regions are used to provide cross-isolateprotection and/or diagnosis. In this instance, the mixture of peptideimmunogens is commonly referred to as a “cocktail” preparation for useas an immunogenic composition or a diagnostic reagent. The peptides ofthis invention can be used in such a peptide.

The peptides of the present invention are useful as antigens inimmunoassays which include but are not limited to enzyme-linkedimmunosorbent assays (ELISA), RIAs, and other non-enzyme linked antibodybinding assays, or procedures known in the art for the detection ofanti-HIV antibodies. For a review of immunological and immunoassayprocedures in general, see Basic and Clinical Immunology 7th Edition (D.Stites and A. Terr, eds.) 1991. Moreover, the immunoassays of thepresent invention can be performed in any of several configurations,which are reviewed extensively in Enzyme Immunoassay, E. T. Maggio, ed.,CRC Press, Boca Raton, Fla. (1980); “Practice and Theory of EnzymeImmunoassays,” Tijssen; and in Antibodies A Laboratory Manual, Harlowand Lane, Cold Spring Harbor Laboratory Press, 1989.

In ELISA assays, for example, the peptides are immobilized onto aselected surface, for example a surface capable of binding peptides,such as the wells of a polystyrene microtitre plate. After washing toremove incompletely adsorbed peptides, a non-specific protein, such as asolution of bovine serum albumin (BSA) or casein, that is known to beantigenically neutral with regard to the test sample may be bound to theselected surface. This allows for blocking of non-specific adsorptionsites on the immobilizing surface and thus decreases the backgroundcaused by non-specific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials to be tested, in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents such as solutions of BSA, bovine gamma globulin(BGG) and/or phosphate buffered saline (PBS)/Tween. The sample is thenallowed to incubate for from about 2 to 4 hours, at temperatures such asof the order of about 25° to 37° C. Following incubation, thesample-contacted surface is washed to remove non-immunocomplexedmaterial. The washing procedure may include washing with a solution suchas PBS/Tween, or a borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound peptides, and subsequent washing, the occurrence, and evenamount, of immunocomplex formation may be determined by subjecting theimmunocomplex to a second antibody having specificity for the firstantibody. If the test sample is of human origin, the second antibody isan antibody having specificity for human immunoglobulins and in generalIgG. To provide detecting means, the second antibody may have anassociated activity, such as an enzymatic activity that will generate,for example, a color development upon incubating with an appropriatechromogenic substrate. Quantification may then be achieved by measuringthe degree of color generation using, for example, a visible spectraspectrophotometer.

Other uses

Molecules which bind to the conserved sequences on which the inventionis based, particularly binding proteins, antibodies, antibody-relatedmolecules and structural analogs thereof, are also of possible use asagents in the treatment and diagnosis of AIDS and related conditions.

For targeted delivery of toxins or other agents, e.g., by use ofimmunotoxins comprising conjugates of antibody to the relevant peptidesand a cytotoxic moiety, for binding directly or indirectly to a targetconserved sequence of, for example, or gp120 or gp41.

For targeted delivery of highly immunogenic materials to the surface ofHIV-infected cells, leading to possible ablation of such cells by eitherthe humoral or cellular immune system of the host.

For detection of HIV, e.g., using a variety of immunoassay techniques.

In yet a further diagnostic embodiment, the peptides of the presentinvention (individually, or as mixtures including cocktail preparations)are useful for the generation of HIV-1 antigen-specific antibodies(including monoclonal antibodies) that can be used to detect HIV-1 orspecific antigens thereof, or to neutralize HIV-1 in samples includingbiological samples.

In an alternative diagnostic embodiment, the peptides of the presentinvention can be used to specifically stimulate HIV-specific B-cells inbiological samples from, for example, HIV-infected individuals.

A further diagnostic embodiment includes the use of the peptides todetermine prognosis for Long Term Non-Progressor patients towards AIDS.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.All references are herein incorporated by reference.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily recognize a variety ofnoncritical parameters which could be changed or modified to yieldessentially similar results.

Example 1 Identification of HIV specific phagotopes

a. Affinity selection of HIV-1 mimotopes

In order to select for B cell epitopes specifically recognized by serumantibodies of HIV-1-infected subjects, random phage libraries (RPLs)displayed on phages were screened with HIV-1 positive sera fromlong-term non-progressor (LTNP) subjects. This population was chosenbecause LTNP subjects show higher titers of neutralizing antibodies thansera from AIDS patients (Montefiori et al., J. Infect. Dis. 173:60(1996)). In order to maximize the detection of HIV-specific peptides,phagotopes were first selected by immuno-affinity purification with theIgG of one HIV-1 positive LTNP individual, and then subjected toimmunoscreening by using a second HIV-1 positive LTNP serum.

More particularly, human sera were collected from HIV-1 positive orHIV-1 negative control subjects. Criteria for definition of long termnon-progression of AIDS were as previously described (Pantaleo et al.,N. Engl. J. Med. 332:209 (1995)). Two peptide libraries composed ofrandom nonamers displayed on the N terminus of pVIII major coat proteinof filamentous phages either unconstrained (pVIII9aa) (Felici et al., J.Mol. Biol. 222:301 (1991)), or flanked by two cysteines (pVIII9aa-cys)(Luzzago et al., Gene 128:5 (1993)) were screened as described (Prezziet al.,. J. Immunol. 156:4504 (1996)).

In the immunoaffinity selection, serum IgG was linked to magneticmicrobeads (tosyl-activated Dynabeads M450; Dynal, Lake Success, N.Y.)previously coated with an anti-human (Fc-specific) polyclonal antibody(goat anti human IgG Fe-specific; Sigma, SL Louis, Mo.) at 200 μg/ml ofbeads suspension. 2×10¹¹ transducing units (TU) of phage particles wereapplied to IgG-coated beads and incubated for 16 h at 4° C. Afterextensive washing, bound phages were eluted with 0.1 M HCl/glycinebuffer pH 2.2 and neutralized.

b. Immunoscreening

The secondary immunoscreening was performed as follows: TG-1 cells wereinfected with eluted phages at a multiplicity of infection (MOI) of1×10⁻³ and plated at a density of 1×10⁴ TU/plate. The following day,bacterial colonies were collected, amplified and superinfected withM13K07 at an MOI of 50. Two thousand colonies were replated on a lawn ofTG-1 cells in the presence of 35 μg/ml ofisopropyl-1-thio-β-D-galactoside (IPTG). Plates were layered withnitrocellulose filters for 16 h at 37° C. Filters were incubated withserum at a 1:50 dilution in immunoscreening buffer (5% non-fat dry milk,0.1% Nonidet p40, 3×10¹¹ wild-type phages, 5×10⁹ M13K07 UV-killedphages/ml, 10 μl of TGI bacterial extract) for 16 h, at 4° C. Positivecolonies were detected by an anti-human (Fc-specific) alkalinephosphatase-conjugated antibody (Sigma).

To validate that the bound phagotopes carry HIV-specific epitopes,positive colonies were tested by ELISA for reactivity with sera frommultiple HIV-infected individuals and counter-screened with anequivalent number of sera from HIV-negative subjects. For the EJISAanalysis, microtiter plates were coated with anti M13 antibody(Pharmacia, Piscataway, N.J.) at 10 μg/ml overnight at 4° C. in coatingbuffer. Fifty μl of cleared phage supernatant with an equal volume ofblocking buffer were incubated for 1 h at 37° C. Plates were washedextensively and supplemented with human serum at 1:100 dilution followedby an overnight incubation at 4° C. After washing, wells were coatedwith an anti-human (Fc-specific) alkaline phosphatase-conjugatedantibody. Plates were washed and developed. Results were expressed asthe difference between OD_(405 nm) and OD_(620 nm) by an ELISA reader.

There were two selections. In selection 1, LTNP sera 6090 and 3976 wereutilized for immunoaffinity and immunoscreening steps, respectively.Similarly, selection 2 was performed by using LTNP sera 3872 and 8075.Selection 1, performed on a cysteine constrained pVIII9aa-cys library(Luzzago et al, Gene 128:5 (1993)), resulted in the identification offive HIV-specific clones; selection 2, conducted on an unconstrainedpVIII9aa library (Felici et al., J. Mol. Biol. 222:301 (1991)) led toisolation of five additional phagotopes (FIG. 1 a).

All the selected clones were found to react with 22 LTNP sera and 25AIDS sera with a recognition frequency (f) ranging from 23 to 64%; allclones tested negative by ELISA with 50 HIV-negative sera. It was highlyunlikely that the frequency distribution of each phagotope between HIVpositive and negative sera could have occurred by chance (p<0.001).Accordingly, the clones were considered HIV-1-specific phagotopes. Eachserum manifested a distinct pattern of reactivity with the pool ofphagotopes (FIG. 1 a). Some sera, such as 8873 and 1276, recognized mostphagotopes indicating a broad antibody specificity, whereas sera 2214,5223, and 8075 reacted with only one phagotope.

Clone p217 was restricted in its reactivity to a subset of LTNP sera (f:23); however, it was completely unreactive with a pool of AIDS sera(FIG. 1 a). This suggests that p217-specific antibodies may exert aprotective role in disease progression. Analysis of the reactivities ofsera for each phagotope showed that antibody titers to p163, p217 andp335 were significantly higher in sera from LTNPs than from AIDSpatients (p<0.05, FIG. 1 b). Again, these results suggest that antibodyresponses to these epitopes might afford a degree of protection againstdisease progression.

Example 2 Characterization of HTV-1 mimotopes

The amino acid sequences of the phage-displayed peptides are shown inFIG. 2. A BLAST analysis revealed that the p195 epitope shares sequencehomology with the gp120 V1 region (residues 112-120) of HIV1-U116374, aprimary isolate from an acute seroconverter (Zhu et al., J. Virol.69:1324 (1995)); the p217 sequence matched with the gp120 C2 region(residues 198-205) of HIV1-U116077, a primary isolate from an AIDSpatient (Shapshak et al., Adv. Exp. Med. Biol., 373:225 (1995)) (FIG. 2a).

Residues within these regions have been predicted to be immunologicallyaccessible by selected mAbs and by X ray crystal structure (Kwong etal., Nature 393:648 (1998); Wyatt et al., Nature 393:705 (1998)).Moreover, the p197 epitope mapped to a region of gp41 (residues 602-605)of the HIVANT170 primary isolate (Vanden et al., J. Virol. 68:1586(1994)). This region is conserved among primary isolates of HIV subtypesA through G and defines a disulfide-bonded structure important for theassociation of gp120 and gp41 (Cotropia et al., J. Acquir. Immune Defic.Syndr. Hum. Retrovirol. 12:221(1996)). Moreover, this domain is proximalto an exposed region of gp41 containing the linear epitope ELDKWArecognized by 2F5, a monoclonal antibody capable of neutralizing amajority of a panel of typical primary isolates identified in the UnitedStates (D'Souza et al., J. Infect. Dis. 175:1056(1997)) (FIG. 2 a,b).Thus, the peptides expressed on p195, p217, and p197 are antigenicmimics of epitopes expressed in primary HIV isolates from subjects atdifferent stages of disease. No sequence homology with HIV proteins wasfound in the remaining clones, suggesting that they may representimmunological mimics of conformational HIV-1 epitopes (FIG. 2 c).

Example 3 Immunoaffinity of antibodies specific to the peptides

The antibody reactivities shown in FIG. 1 a indicate that thephage-displayed peptides behave as antigenic mimics of viraldeterminants generated in the course of HIV infection. Therefore, it ispossible to immunoaffinity purify antibodies specific for each phagotopefrom sera of HIV-infected individuals by using single phagotopes asligands. To this end, p195, p197, p217, p287 and p335 were utilized topurify the phagotope-specific antibodies from LTNP serum 6090.

Affinity purification of phagotope specific human antibodies wasaccomplished using 60 mm diameter dishes coated with 5×10¹¹CsCl-purified phages overnight at 4° C. After washing and blocking,human serum (1:100 dilution) was added and incubated for 16 h at 4° C.After extensive washing, bound antibodies were eluted with glycine-HClbuffer pH 2.2. Antibody concentration was determined by an in-houseELISA with a low detection level of 1-2 ng/ml.

The phagotope specific antibodies, purified from serum to IgGconcentrations of 5-10 ng/ml, recognized HIV-1 proteins by ELISA (FIG. 3a); this reactivity was specifically displaced by the relatedphagotopes, but not by wild-type or unrelated phages (FIG. 3 b and datanot shown). Moreover, peptides corresponding to the epitopes displayedon phages p195, p197, and p335 effectively displaced the binding of Absto HIV-1, indicating that these peptides acquire in solution aconformation similar to the one expressed by both the phage-displayedpeptides and the HIV epitopes (FIG. 3 c). Only a partial competition wasobserved in the case of peptide 217 and 287, indicating that expressionof these peptides on the surface of the phages is essential to acquire aconformation mimicking HIV epitopes (FIG. 3 c), as previously suggested(Meola et al., J. Immunol., 154:3162 (1995)).

When the phagotope-specific antibodies were tested in immunoblotting, adistinctive reactivity was found. Ab-195 and Ab-217 recognized gp160 andgp120, consistent with the mapping of these epitopes at enveloperegions. In addition, Ab-287 and Ab-335 also detected HIV-gp160 andgp120, indicating that they recognized envelope-specific epitopes (FIG.3 d). No bands were detected by p197-specific antibodies, indicating anintrinsic inability of these antibodies to bind to the cognate epitopeunder denaturing conditions (FIG. 3 d).

Example 4 HIV-1 mimotopes react specifically with sera of SHIV-infectedmonkeys

Simian HIV [SHIV] recombinant viruses expressing HIV env on the backboneof SIV isolates are a useful model of HIV-1 infection in primates(Shibata el al, J. Virol. 71:8141 (1997)). SHIV-infected monkeys raisehigh titers of neutralizing Abs that correlate with long-lastingprotection from subsequent challenge with pathogenic SHIV (Igarashi etal., J. Gen. Virol. 78:985 (1997)) or SIV-mac239 (Miller er al, J.Virol. 71:1911 (1997)). Since the HIV-specific phagotopes areimmunogenic mimics of HIV-1 env proteins (FIG. 1-3), they should berecognized by antibodies of SHIV-infected animals.

In order to test this hypothesis, sera of nine SHIV-infected monkeys andof four uninfected control animals were tested for ELISA reactivity withthe pool of HIV mimotopes. As in the case of HIV-1 infected subjects(FIG. 1 a), sera of SHIV-infected monkeys recognized the HIV-1phagotopes with variable frequencies (FIG. 4). As previously noted,phagotopes p32, p54, and p689 did not match any HIV sequences in thedatabase (FIG. 2 c). The fact that certain SHIV sera recognized thesephagotopes suggests that they are conformational mimics of discreteregions of gp 160, Nef or Tat, since these are the only HIV-specificsequences within SHIV.

Phagotope p 163 and p483, which were consistently recognized by LTNP andAIDS sera (FIG. 1 a), did not react with SHIV sera, suggesting that theyare antigenic mimics of HIV-1 epitopes encoded for gag or pol genes.Sera from uninfected monkeys tested negative in ELISA (FIG. 4). Theseresults indicate that macaques are genetically similar to humans inantigen processing and presentation, since their antibodies efficientlyrecognized human B-cell epitopes; in this regard, the selectedphagotopes should induce HIV-specific antibodies, and could be exploitedto immunize monkeys before SHIV challenge.

Example 5 Production of neutralizing antibodies

HIV-1 mimotopes induce neutralizing antibodies in mice

The HIV-1-binding antibodies of this invention exert neutralizingactivity in vitro if directed to accessible epitopes of infectiousvirions. As antigenic mimics of HIV-1 epitopes, HIV-1 phagotopes have aconformation that fits in the antigen-binding site of the related serumantibodies, and would be expected to elicit antibodies in vivo withspecificities similar to the original serum IgG utilized to select them.To establish the neutralizing effects of the peptides of this invention,HIV-1 phagotopes p195, p197, p217, p287 and p335 were used to immunizeBALB/c or C57B/6 mice.

Phage were CsCl purified and used at a concentration of 6×10¹²particles/ml in 0.9% NaCl with an equal volume of CFA or IFA. Four tofive-week-old female BALB/c and C57B1 mice were immunized by i.p.injection of 200 μl of antigen emulsion at weeks 0, 3, 6, 9, 12 and bledon day 0 and day 7-10 after each additional injection. Serum IgG waspurified from mouse sera with T-Gel Adsorbent (Pierce, Rockford, Ill.).

All mice developed comparable titers of Abs against wild-type phages,and a strong antibody response to the original phagotopes used asimmunogens (not shown). To determine the neutralizing effects of theantibodies, neutralization of HIV_(IIIB) and NL4-3 molecular clones wasmeasured in a MT-2 assay (Montefiori et al. J. Clin., Microbiol. 26:231(1988)). Briefly, cell free virus (500 TCID₅₀/ml) and serial dilutionsof mouse IgG were incubated in triplicate at 37° C. for 1 h before theaddition of MT-2 cells (5×10⁴/well). At 6-8 days post infection,neutralization was quantified by staining viable cells with neutral redfollowed by colorimetric determination of uptake at 540 nm. In the caseof AD8, neutralization assay was performed on PHA-activated PBMC asdescribed (Montefiori et al., J. Virol. 72:1886 (1998)).

Purified IgG from mice immunized with HIV-1 phagotopes exerted asignificant inhibition of infection by HIV1_(IIIB) and NL4-3 isolatesover a wide range of IgG concentrations in an in vitro acute infectionsystem, with 50% protection observed at IgG concentrations of 0.8 to 3μg/ml in neutralization assays with HIV_(IIIB) or NLA4-3, respectively(FIG. 5 a,b). Consistent levels of viral neutralization were alsoobtained in the case of the AD8 primary isolate, with the exception ofp335-specific Abs, which exerted partial protection only at the highestconcentrations (FIG. 5 c).

The selected phagotopes fulfilled the requirement for an effectiveimmunogen. In fact, Abs from phagotope-immunized mice neutralized HIV-1strains in vitro, suggesting that they bind well to the virus underphysiologic conditions and could possibly prevent or inhibit HIVinfection when induced in phagotope-immunized primates. In support ofthis possibility, serum Abs of SHIV-infected monkeys showed a strongreactivity with the phage-displayed epitopes. In addition,bacteriophages are excellent immunogens that induce a specific T celldependent antibody response by parenteral as well as oral administration(Galfre et al., Methods Enzymol., 267:109 (1996); Delmastro et al.,Vaccine 15:1276 (1997)).

Example 6 Immunogenicity of epitopes in Rhesus macaque monkeys

An immunization trial of a cohort of Rhesus macaque monkeys wasinitiated to verify the immunogenic properties of the HIV-1 specificepitopes selected by screening the Random Peptide Libraries displayed onphage as described in the examples above and in J. Immunol. 162:6155(1999). To this end, three groups of five animals were immunized with apool phage 195, 197, 217, 287, and 335 as follows:

Immunization Schedule: 1 injection every 6 weeks.

-   Group I: Wild-type (WT) phage (2.5×10¹⁴ phage particles each    animal/four animals total)-   GroupII φ195; φ197; φ217; φ287; φ335 (5×10¹³ phage particles each    animal/five animal total)-   Group III φ195; φ197; φ217; φ287; φ335 (1×10¹³ phage particles each    animal/five animals total)

The adjuvant QS21 (Aquila Pharmaceuticals) was used in an amount of 100μg/injection. The immunogens were injected intramuscularly at multiplesites. The plasma collected at the 4th injection (the third boost) wastested for the presence of an antibody response specific for the epitopeutilized as the immunogen. Sera from either WT-phage-immunized orepitope-immunized monkeys were tested by ELISA for binding to peptidesrepresenting the original phage-displayed epitopes. As shown in FIG. 6a, sera from monkeys immunized with HIV-epitopes showed a substantialdegree of ELISA reactivity to the different epitopes, while monkeysimmunized with wt phage did not show any specific antibody response(FIG. 6 b). These data indicate that the phagotopes originally selectedfrom RPL are immunogenic in non-human primates and elicited a specificantibody response.

Because the selected epitopes are mimotopes of discrete regions of theHIV-1 envelope, the monkey antibodies would be expected to bind to HIV-1envelope. To test this possibility, the sera collected from theimmunized monkeys were tested by ELISA for binding to HIV-IIIB gp 160.As shown in FIG. 7, serum antibodies from three animals tested positivein ELISA. The above results indicated that epitopes selected from RPLfor their binding to antibodies of HIV-positive subjects function asantigenic mimics of discrete regions of HIV-1 proteins.

Example 7 Engineered peptide epitopes

The peptide sequences present on phagotopes 195, 197, 217, 287 and 335were also modified to obtain peptide epitopes capable of functioning asantigenic and immunogenic mimics of the original epitopes displayed onthe phages. The modifications were generated by randomly changing thenumber of residues flanking the core sequence previously identified,and/or by subjecting the core sequence to amino acid substitutions untilthe resulting peptides were found to exhibit ELISA reactivities similarto that of the original peptides displayed on the phagotopes. The aminoacid sequences of modifed peptides generated in this manner are asfollows:

-   P195 analog: EGEFCKSSGKLISLCGDPAK (SEQ ID NO: 14)-   P197 analog: EGEFCQTKLVCFAAAGDPAK (SEQ ID NO:15)-   P217 analog: EGEFCCNGRLYCQPCGDPAK (SEQ ID NO:16)-   P287 analog: EGEFCCAGQLTCSVCGDPAK (SEQ ID NO: 17)-   P335 analog: CSGRLYCHESWC (SEQ ID NO:18)-   P54 analog: TKTLIYQGA (SEQ ID NO:19)    The engineered eptiopes are then determined to be immunogenic as    described in Example 4-6.

Taken together, our results as demonstrated in the Examples indicatethat a collection of HIV-1 mimotopes can be retrieved from combinatorialphage libraries by taking advantage of the specific antibody repertoireinduced by natural infection, and thus are useful in the development ofeffective HIV-1 vaccines.

1. An antigenic peptide of less than 100 amino acids having an antigenicsubsequence comprising X-KSSGKLISL-X (SEQ ID NOS: 1 and 31), wherein Xis independently an amino acid or sequence of amino acids with theproviso that X is not identical to the amino acid or amino acidsnaturally flanking the subsequences in human immunodeficiency virus-1(HIV-1).
 2. An antigenic peptide of claim 1, wherein the antigenicsubsequence is EGEFCKSSGKLISLCGDPAK (SEQ ID NO: 14).
 3. A peptide ofclaim 1, wherein the antigenic subsequence comprises an antigenicdeterminant that does not give rise to HIV-1-specific antibodies to morethan twelve other antigenic determinants on HIV-1.
 4. A method forraising antibodies against HIV-1, said method comprising administeringto an animal an amount of an antigenic peptide having an antigenicsubsequence comprising X-KSSGKLISL-X (SEQ ID NOS: 1 and 31), wherein Xis independently an amino acid or sequence of amino acids with theproviso that X is not identical to the amino acid or amino acidsnaturally flanking the subsequences in HIV-1, and further wherein thecomposition does not give rise to_HIV-1-specific antibodies to more thantwelve other antigenic determinants on HIV-1, said amount sufficient toraise antibodies in the animal.
 5. The method of claim 4, wherein theantigenic subsequence is EGEFCKSSGKLISLCGDPAK SEQ ID NO: 14).
 6. Amethod for detecting HIV-1-specific antibodies in a person suspected ofbeing infected with HIV-1, said method comprising the step of incubatinga biological sample from the person with an antigenic peptide having anantigenic subsequence comprising X-KSSGKLISL-X SEQ ID NOS: 1 and 31) inan amount sufficient to detect the presence of antibodies which bind tothe antigenic determinant and determining the binding of the antibody tothe antigenic determinant.
 7. The method of claim 6, wherein theantigenic subsequence is EGEFCKSSGKLISLCGDPAK (SEQ ID NO: 14).